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Originally published as Ann Surg Oncol Early Release 10.1245/ASO.2004.03.023 on November 12, 2004

Annals of Surgical Oncology 11:988-997 (2004)
© 2004 Society of Surgical Oncology
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ORIGINAL ARTICLES

Detection of Melanoma Cells in the Lymphatic Drainage after Lymph Node Dissection in Melanoma Patients by Using Two-Marker Reverse Transcriptase-Polymerase Chain Reaction Assay

Ruka Wlodzimierz, MD, Piotr Rutkowski, MD, Zbigniew I. Nowecki, MD, Jadwiga Kulik, MD, Anna Nasierowska-Guttmejer, MD and Janusz A. Siedlecki, PhD

From the Departments of Soft Tissue/Bone Sarcoma and Melanoma (RW, PR, ZIN), Molecular Biology (JK, JAS), and Pathology (AN-G), M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland.

Correspondence: Address correspondence and reprint requests to: Piotr Rutkowski, MD, Department of Soft Tissue/Bone Sarcoma and Melanoma, M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, W. Roentgena Str. 5, 02-781, Warsaw, Poland; Fax: 48-22-643-9791; E-mail: rutkowskip{at}coi.waw.pl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: The aim of this study was to evaluate the role of melanoma gene expression as a marker of the presence of melanoma cells in lymphatic drainage routinely collected after lymphadenectomy and to correlate reverse transcriptase-polymerase chain reaction (RT-PCR) assay results with recurrence, survival, and prognostic factors.

Methods: We collected 24-hour postoperative lymphatic drainage samples (between days 2 and 4) from 93 patients with stage III melanoma who underwent radical lymphadenectomy between May 2002 and November 2003. We used RT-PCR assays with primers specific for the tyrosinase and MART-1 (Melan-A) genes. The samples were considered positive if at least one marker was expressed. Median follow-up time was 12.8 months.

Results: In 18 (19.4%) of 93 patients, the RT-PCR assay results were positive: in 8 of 18 for tyrosinase only, in 7 of 18 for MART-1 only, and in 3 of 18 for both markers. We observed a significantly higher recurrence rate in patients with positive RT-PCR results (15 of 18; 83%) than negative results (26 of 75; 35%; P = .0001). Positive results of RT-PCR correlated with the number of involved lymph nodes (P = .0001) and extracapsular extension of nodal metastases (P = .03). We observed significant differences in overall and disease-free survival for RT-PCR–positive and –negative patients in univariate and multivariate analyses.

Conclusions: We observed positive RT-PCR assay results for melanoma cells in the lymphatic drainages of approximately 20% of patients after lymphadenectomy. This correlated significantly with early recurrence and shorter survival. These results may suggest that the RT-PCR assay could be useful for routinely analyzing postoperatively collected lymphatic drainage in stage III melanoma patients and for predicting disease progression.

Key Words: Melanoma • Lymph node • Metastasis • RT-PCR assay • Prognostic markers


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The frequency of cutaneous melanoma has been increasing recently. In cases with regional lymph node involvement, the risk of distant metastases is particularly high. Many efforts have been undertaken to design sensitive and specific molecular methods investigating characteristic melanoma markers. The detection of circulating tumor cells in the course of reverse transcriptase-polymerase chain reaction (RT-PCR) assays may allow identification of cases with worse prognosis or may offer a surrogate marker of residual disease.1–4 However, the value of RT-PCR assay results for melanoma gene expression in peripheral blood and lymph nodes is still controversial. It has been suggested that the ratio of patients with positive assay results would correlate with the ratio expected to have disease recurrence.3,5,6 Other authors do not report any correlation between melanoma stage or prognosis and RT-PCR blood tests.7–11 The most intensively investigated marker analyzed in RT-PCR tests is tyrosinase (EC 1.14.18.1)—a key enzyme involved in melanin synthesis that is transcribed actively mainly in melanocytes. This technique allows detection of a single melanoma cell in 1 mL of blood (1 tumor cell per 106–107 normal cells). However, other cells, including Schwann cells, may also express this, presumably, tissue-specific marker of melanocyte detection. Moreover, in a number of advanced cases, melanoma cells lose tyrosinase expression. A meta-analysis of RT-PCR assays for tyrosinase messenger RNA (mRNA) as a marker of circulating cutaneous melanoma cells performed by Tsao et al.12 has shown the limited value of this assay and the lack of reliable data on the outcome of stage I to III patients in whom positive results were obtained with this test. Thus there is a need to search for other melanoma markers, such as p97, Melan-A/MART-1, MUC18, uMAG, and gp100, and to combine them in multiple-marker tests.13–15 We have previously published the results of a study on a two-marker RT-PCR assay with tyrosinase and MART-1 (melanoma antigen recognized by T cells) in the detection of circulating melanoma cells in peripheral blood.16 MART-1 is frequently expressed (>85%) by melanoma tumor cells and melanoma cells lines, but it is not expressed in nonmelanoma malignancies or in healthy individuals. The addition of MART-1 analysis improves the sensitivity of melanoma cell detection.16–20 It has also been reported that the sensitivity and specificity of PCR with MART-1 is similar to that with tyrosinase, but it seems to identify a different subgroup of patients—one with locoregional rather than distant metastases.21

Regional lymph node involvement is one of the most important factors negatively affecting the clinical outcome of patients with cutaneous melanoma.22–24 The 5-year survival rate decreases, on average, to <50% when regional lymph node metastases are found. Subjects with localized disease and without nodal metastases (stage I/II) have a relatively good prognosis, whereas those with disseminated disease (stage IV) are of low clinical interest regarding their predictive factors because of fatal outcomes and the lack of effective treatment. Patients with lymph node metastases are the most important therapeutic problem in melanoma, and they are the target for experimental adjuvant therapy. However, this group consists of patients with a varied prognosis (the 5-year survival rate ranges from 24% to 69%).22,23 Thus, the main issue is to set the criteria that would precisely identify patients at high risk of dissemination and unfavorable outcome and who may benefit from adjuvant experimental treatment. Analyses of blood samples from stage III patients have revealed remarkable differences in tyrosinase-positive rates (0%–86%).9,11,25–28 The blood RT-PCR tests provide contradictory and questionable results for patient prognosis.6,25

We hypothesized that evaluation of occult melanoma cells in lymphatic drainage obtained after therapeutic lymph node dissection in melanoma patients with nodal metastases may allow identification of subjects with a high or low risk of relapse. In our prospective study, we investigated the presence of melanoma cells in routinely collected postoperative lymphatic drainage collected from stage III patients after therapeutic regional lymph node dissection. We used a highly sensitive two-marker (tyrosinase and MART-1) RT-PCR assay established in our laboratory.24 The primary aim of the study was to investigate the relationship between RT-PCR assay results for melanoma-specific genes, early disease recurrence, and patient survival, as well as established prognostic factors for melanoma. We believe that tyrosinase and MART-1 may form a useful marker combination in RT-PCR assays for the detection of melanoma cells in postoperative lymphatic fluid and that the results of this assay may correlate with early disease recurrence and patient survival.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Between May 2002 and November 2003, 93 consecutive patients with histologically proven cutaneous melanoma and regional nodal involvement (stage III according to the American Joint Committee on Cancer [AJCC] 2002 classification22) underwent therapeutic lymphadenectomy at the Department of Soft Tissue/Bone Sarcoma and Melanoma of the M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology in Warsaw, Poland. In 41 patients the original procedure consisted of lymphatic mapping and, in case of positive sentinel lymph nodes, subsequent complete sentinel lymph node dissection (SLND group). Fifty-two patients underwent radical lymphadenectomy because of clinically detected (palpable) regional lymph node metastases, confirmed pathologically by fine-needle aspiration biopsy (completion lymph node dissection [CLND] group). Each patient provided informed consent. The study had been approved by the local bioethics committee according to Good Clinical Practice guidelines. Patients were enrolled onto the study only when they met the following criteria.

Inclusion criteria were as follows:

  1. Stage III melanoma (TxN1 to N3M0) with regional nodal metastases (all histological diagnoses were confirmed by the Department of Pathology).
  2. Radical axillary or ilioinguinal lymphadenectomy was performed.
  3. Age >18 years.
  4. World Health Organization performance status of 0 or 1.
  5. No distant metastases.

Exclusion criteria were as follows:

  1. Diagnosis of noncutaneous melanoma.
  2. Diagnosis of other malignancies.
  3. Pregnant or lactating women.
  4. Distant metastases.
  5. Lack of patient consent.

The patients had not undergone any other preliminary selection.

The lymphatic drainages (minimal volume, 50 mL) were collected for 24 hours (routine suction). The collection was held for 48 to 96 hours after radical lymphadenectomy. The clinicopathologic stage of disease was determined by pathologic evaluation of the primary lesion and of the dissected lymph nodes, as well as by physical examination and routine imaging examinations (chest x-ray and ultrasonography of the abdominal cavity).

In 41 analyzed patients, we performed preoperative lymphoscintigraphy combined with intraoperative vital blue dye (Patent Blau V®), lymphatic mapping, and intraoperative lymphoscintigraphy with a handheld gamma-detecting probe (Neoprobe 1000®; Neoprobe Corp., Dublin, OH, or Navigator®; RMD, Watertown, MA). During routine pathologic examination, sentinel lymph nodes were cut serially along the major axis and stained with hematoxylin and eosin. Paraffin-embedded specimens were examined with light microscopy (x40 and x200). In doubtful cases, additional immunohistochemical staining (S-100 and HMB-45) was performed. For the purposes of this study, all positive and false-negative sentinel lymph nodes were reviewed by the same blinded pathologist.

Patient characteristics are listed in Table 1. The median Breslow thickness of the primary tumor was 5.0 mm (range, .8–21.0 mm). Many patients (64.5%) presented with ulceration of the primary lesion (according to the AJCC criteria, defined as "absence of an intact epidermis overlying a major portion of the primary melanoma based on microscopic examination of the epidermis," and the stratum corneum of the epidermis should not be intact). In the CLND group, two patients presented with an occult primary lesion. In 48 patients (51.6%) we performed radical axillary lymph node dissection, and in 45 patients (48.4%) we performed radical ilioinguinal lymphadenectomy (routinely superficial and deep levels below the inguinal ligament to the level of the aortic bifurcation, combined with obturatory lymph node dissection). Final pathologic examination after lymphadenectomy revealed extracapsular extensions of melanoma cells in 49.5% of cases with involved lymph nodes (defined as metastatic melanoma cells in adjacent soft tissue with any histological evidence of breach of the node capsule).


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  		TABLE 1. Patient characteristics and distribution of clinicopathologic features in patients from the RT-PCR–positive and –negative groups
 
Postoperative follow-up consisted of physical examination and routine imaging investigations (chest x-ray and ultrasonography of the abdominal cavity). Thorough examination (every 3 months during the first 2 years) was routinely recommended.

In the case of one patient who had undergone synchronous bilateral axillary dissection, the numbers of lymph nodes, metastases, and pathologic features were summed for statistical analysis. The median follow-up time was 12.8 months for survivors (range, 6–24.5 months).

Synthetic Oligonucleotides
All primers were synthesized by using an Applied Biosystems apparatus type 391. The primer sequences used in this work are listed in Table 2.


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  		TABLE 2. Sequences for polymerase chain reaction primers
 
Lymph Fluid Preparation and RNA Isolation
The lymph fluid samples (50–100 mL) were centrifuged (500 x g; 10 minutes) immediately after collection. The pellet was collected and incubated for 10 minutes on ice with red blood cell lysing solution.

RNA was prepared from fresh or frozen pellet according to the method presented by Chomczynski and Sacchi.29 RNA was further purified from trace amounts of DNA according to the method recommended by the DNase I (ribonuclease free) manufacturer (Gibco-BRL), although the amount of DNase I used was four times smaller. DNase I was inactivated by a 10-minute incubation at 65°C and phenol extraction. RNA concentrations were determined spectrophotometrically. RNA from the human melanoma cell line MeW151 and normal and fetal fibroblast lines was directly isolated as described by Chomczynski and Sacchi.29 Before complementary DNA (cDNA) synthesis, each RNA sample was examined in the course of electrophoresis on a denaturing agarose gel. We did not observe any significant degradation of the 28S and 18S RNA bands (data not shown). The quality of the cDNA was controlled with PCR by using primers for human glyceraldehyde-3-phosphate dehydrogenase 1 and 2 (Table 2).

RT-PCR Methods
The RT-PCR assay was performed as described previously.16 For reverse transcription we used 2 µg of RNA. RNA was incubated at 65°C for 10 minutes and then put on ice. The solution was supplemented with the remaining RT reagents and SuperScript reverse transcriptase to a final volume of 20 µL, as recommended by the manufacturer (Gibco-BRL). The reaction mixture was incubated at 37°C for 1 hour. The cDNA product was further purified by phenol/chloroform extraction and precipitation with 2.5 volumes of ethanol. The precipitated pellet was dissolved in 15 µL of diethyl pyrocarbonate–treated water. The reaction mixture for the first round of PCR contained, in a final volume of 25 µL, 1x PCR buffer, 200 µM of each deoxynucleoside triphosphate, 1.5 mM MgCl2, 50 ng of primers (Table 2),16,30,31 5 µL of purified cDNA solution, and 2.5 U of Taq polymerase. Before amplification, the samples were heated to 94°C for 2 minutes. The amplification conditions were 94°C for 45 seconds, 60°C for 45 seconds, and 72°C for 45 seconds for 30 cycles. Amplification was finished by a 10-minute incubation at 72°C. The reamplification with nested primers was performed under the same amplification conditions (except for the second round of amplification with MART1 nested primers, for which the magnesium concentration was increased to 2 mM and the annealing temperature was decreased to 55°C) for another 30 cycles by using .5 µL of the product of the first round of PCR as a template. The resulting reamplification products (10 µL) were analyzed on 2% agarose with pUC19 digested with DdeI restriction endonuclease as a molecular weight standard.

The drain fluid sample was assumed to be positive if at least one marker was expressed. The people performing the PCR assay were blinded to disease status, and those recording the disease status were blinded to the PCR assay results.

Statistical Analyses
All statistical analyses were performed with Statistica software (StatSoft, Tulsa, OK). Contingency tables were analyzed by the {chi}2 test. Groups were compared for age differences by using the t-test for normal distribution of parameters.

Overall survival (OS) was calculated from the date of radical lymph node dissection to the date of the most recent follow-up or death. Similarly, disease-free survival (DFS) was estimated from the date of lymphadenectomy to the date of the most recent follow-up or disease recurrence. Follow-up ended on May 21, 2004. For survival analysis, we used the Kaplan-Meier method in combination with the log-rank test for univariate analysis and the Cox proportional regression hazard model for multivariate analysis. Differences were considered statistically significant if P values were <.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RT-PCR assay results were positive in 18 (19.4%) of 93 patients: in 8 cases for tyrosinase only, in 7 for MART1 only, and in 3 for both markers. Positive RT-PCR results correlated with the established predictive factors for nodal involvement: the number of metastatic regional lymph nodes (P < .0001) and extracapsular extension of lymph node metastases (P = .03) (Table 3). Positive results of this assay were found only in 4 (9.7%) of 41 patients after SLND, as compared with 14 (26.9%) of 52 CLND patients (P = .03).


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  		TABLE 3. Patterns of first relapse in patients with positive and negative RT-PCR assay results after radical lymphadenectomy
 
We observed a significantly higher rate of recurrence in patients with positive RT-PCR results (15 of 18 cases; 83.3%) than in patients with negative results (26 of 75 cases; 35%; P = .0001). Patients with positive RT-PCR results demonstrated the following pattern of disease progression: in-transit metastases/local recurrences (n = 4), regional lymph node recurrence (n = 1), lung metastases (n = 3), liver metastases (n = 3), soft tissue metastases (n = 3), and brain metastases (n = 1). They did not differ from the characteristics of progression in patients with negative RT-PCR results for lymph fluids (Table 3).

Survival Analysis
The median OS of the entire group of patients, calculated from the date of radical lymphadenectomy, has not yet been reached, and the estimated 18-month OS rate was 63.5%. The estimated 18-month OS rates for the RT-PCR–positive and –negative groups were 23% and 76%, respectively (Fig. 1), and there were significant differences between groups (P = .001). Twenty-one patients died of melanoma during follow-up, and 10 of these (48%) had at least 1 positive RT-PCR tumor marker in the lymphatic fluid specimens. Conversely, 72 patients were alive at the end of the study, but in only 8 of these cases (11%) did we find positive melanoma markers in the lymphatic drainage (P = .0002).



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  		FIG. 1. Overall survival time for reverse transcriptase-polymerase chain reaction (RT-PCR)–positive and –negative groups (calculated from the date of radical lymphadenectomy).

 
The median DFS time was 13.3 months, and the estimated 18-month DFS rate was 49.5%. There were significant differences in the DFS (calculated from the date of lymphadenectomy) of RT-PCR–positive and –negative patients: the 18-month DFS rate for the RT-PCR–positive group was 10%, and that for the RT-PCR–negative group was 55% (P = .0001; Figure 2).



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  		FIG. 2. Disease-free survival time for reverse transcriptase-polymerase chain reaction (RT-PCR)–positive and –negative groups (calculated from the date of radical lymphadenectomy).

 
Univariate Analysis
The following factors were found to have a significant effect on OS: extracapsular tumor extension in case of involved lymph nodes (P = .001), the number of metastatic lymph nodes (P = .001), patient sex (P = .02), primary melanoma thickness (P = .05), and the results of the lymph RT-PCR assay (P = .0001). No significant correlations were found between OS and patient age, primary tumor site, Clark level, regional lymphatic basin site, type of metastasis (SLND vs. CLND), primary tumor ulceration, and pathologic subtype of the primary lesion. In the univariate analysis, the presence of at least one RT-PCR tumor marker correlated significantly with decreased patient survival (Fig. 1).

The following factors significantly influenced DFS: the number of metastatic lymph nodes (P = .004), the type of metastasis (SLND vs. CLND; P = .02), the presence of extracapsular invasion in involved lymph nodes (P = .002), and the results of lymph RT-PCR assay (P = .0001; Fig. 2). No significant correlation was found between DFS and patient age, sex, primary tumor ulceration, primary tumor thickness, primary tumor Clark level, subtype of primary lesion, primary tumor site, or regional lymphatic basin site.

Multivariate Analysis
The following factors were found to be independent predictors of poor OS in stage III melanoma patients: nodal extracapsular melanoma invasion (P = .03) and positive RT-PCR assay results (P = .01) (Table 4). In the multivariate analysis, only positive RT-PCR assay results (P = .0004) correlated independently with poorer DFS (Table 5).


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  		TABLE 4. Predictive factors influencing the overall survival of the entire group of patients according to multivariate analysis
 

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  		TABLE 5. Predictive factors influencing the disease-free survival of the entire group of patients according to multivariate analysis
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ever since Smith et al.1 reported the possibility of detecting melanoma cells circulating in the blood by RT-PCR assay, many researchers have investigated this issue to provide early detection of melanoma cells in blood, lymph nodes, and bone marrow for both diagnostic and prognostic purposes.32–35 The value of RT-PCR for the detection of circulating tumor cells is, at present, unclear because of conflicting reports. These inconsistencies may arise from differences in methodology, as well as from poor understanding of the biological significance of circulating tumor cells. Positive results of RT-PCR tests for melanoma molecular markers are more common in advanced stages of the disease (stage IV) and, because of fatal outcomes, have a low clinical predictive value. Similarly, patients in stage I/II with negative results of sentinel lymph node biopsy (performed routinely in our center24) have a rather good prognosis, and in their case, the less frequently positive results of RT-PCR do not offer significant additional data. There are few reports concerning molecular marker detection in the blood of patients with stage III melanoma. Osella-Abate et al.25 report tyrosinase expression in the blood of these patients to be an independent variable of DFS, whereas Mellado et al.36 and Gogas et al.37 have found that tyrosinase expression in clinically melanoma-free patients treated with adjuvant interferon correlates with shorter DFS. Also, Wascher et al.14 demonstrated a relationship between multiple-marker RT-PCR blood assay results and decreased survival in stage III patients receiving melanoma vaccines. Conversely, Reinhold et al.8 have published contradictory results. They did not discover any use of tyrosinase-specific mRNA detection in blood samples for the detection of tumor progression. The results of other studies38,39 suggest that peripheral blood multimarker RT-PCR assays fail to predict recurrence in stage III melanoma. Palmieri et al.40 suggest that circulating melanoma blood cells and their markers do not provide prognostic data in addition to the stage of disease in multivariate models and should not be applied in clinical practice. The possible explanation for these differences may be that the amounts of target mRNA (more than 10-fold below the detection threshold of the assay) are low,41–43 and the persistence of melanoma cells in peripheral blood is transient. The reliability of molecular blood tests may be improved by increasing the volume of blood samples (which is ethically unacceptable) and by multiple testing of the same patient during follow-up. Serial RT-PCR diagnostic testing of multiple, repeated peripheral blood samples may allow prediction of melanoma progression more accurately,37,40,41 but it is troublesome and costly. Also, the technique of immunobead filtration may help to solve this problem. Additionally, some melanoma cells cannot express investigated markers. The combination of two or more molecular melanoma markers for testing allows minimization of the mistakes associated with undifferentiation and heterogeneity of melanoma cells and increases test sensitivity.

The results of molecular investigations into the presence of melanoma in lymph nodes are more encouraging,44–46 but more extensive studies are necessary to evaluate this procedure. With the RT-PCR technique, additional positive sentinel nodes are found in 36% to 55% of cases as compared with immunohistochemistry47–51; however, false-positive results for tyrosinase have also been reported (nevocytes and Schwann cells may express tyrosinase mRNA or tyrosinase and may survive in macrophages). The prognosis of patients with PCR-positive but histology/immunohistology-negative sentinel nodes is now reported to be worse than that of patients in whom the results are negative according to both techniques.52 The multiple melanoma marker panel is used for the evaluation of sentinel lymph nodes in a currently conducted multicenter randomized clinical trial—the Sunbelt Melanoma Trial.53 However, until all the doubts are cleared, it is difficult to recommend this method for routine use.

In this study we have focused our attention on patients with surgically treated high-risk locoregional disease—regional lymph node metastases. At present, the only standard treatment in these patients is radical surgical lymph node dissection of the involved basin. These intermediate-stage patients are major candidates for adjuvant treatment trials. Unfortunately, the results of these adjuvant treatment options are disappointing. This group of patients (stage III according to the 2002 AJCC staging system) is heterogenous with respect to the risk of mortality; it cumulates cases with different prognoses. Their probability of 5-year survival ranges from 24.0% to 69.5%.22,23 No validated clinical assays are capable of providing accurate and early identification of patients who may experience disease recurrence or die of their disease. This would be important for designing future adjuvant therapy trials, because patients at high risk of ultimate treatment failure may be considered for aggressive treatment protocols before the onset of clinically detectable metastases. The results of such an assay may influence treatment plans.

The basic aim of this study was to analyze the usefulness of the two-marker RT-PCR assay for detection of melanoma cells in routinely collected lymphatic drainage fluid after therapeutic lymphadenectomy in melanoma patients with nodal metastases (detected through sentinel lymph node biopsy or clinically detected and confirmed pathologically by fine-needle aspiration biopsy) and without distant metastases. Our hypothesis was that melanoma markers in lymphatic drainage after radical nodal dissection may serve as a surrogate of subclinical residual disease and may suggest further recurrences. This may be particularly attractive in stage III melanoma and for detecting the spread of tumor cells through lymphatic vessels. We have analyzed the relationship between RT-PCR–positive results and the clinical course of the disease to attempt to prove that melanoma cell detection in the lymph is associated with a higher relapse rate and shorter survival. The tests were performed on the lymphatic drainage fluid, which is routinely collected during the postoperative period in patients undergoing radical lymph node dissection. This method does not burden the patients and allows testing large volumes of lymph fluid (usually more than 50 mL over 24 hours). The presented technique has many advantages over testing blood samples: it allows analysis of larger samples, which is especially beneficial when low concentrations of target cells are expected, and it avoids the limitations caused by the intermittent nature of melanoma cell shedding.

We demonstrated melanoma-associated mRNA markers as surrogates of melanoma cell presence in 19.4% of lymphatic drainages after therapeutic lymphadenectomy. Of the 18 patients who had at least 1 positive marker, i.e., were considered melanoma cell positive, 15 (83%) relapsed, as compared with 26 recurrences (35%) in the RT-PCR–negative group of patients. We found that positive results for at least one mRNA marker in lymphatic fluid collected after radical lymphadenectomy correlated significantly with a higher risk of relapse, as well as with DFS and OS. These significant relationships between RT-PCR test results and the course of disease are reflected in the relatively short follow-up time (<2 years). Most recurrences in stage III melanoma occur within 2 to 5 years after lymphadenectomy. Longer follow-up time in a larger cohort of patients is necessary to assess the value of this test. However, it seems that the evident differences in DFS observed between RT-PCR–positive and –negative patients, even during the short follow-up time, are valuable especially when the end point of DFS consists of the combination of local recurrences, metastases, and survival. We have also demonstrated that RT-PCR results correlate with the pathologic features of metastatic lymph nodes and such well-established prognostic factors as the number of involved nodes and extracapsular nodal invasion.

The detailed biological significance of our findings is still poorly understood. The risk of relapse is higher in patients with positive test results. Negative RT-PCR results do not exclude tumor progression; 26 patients with negative RT-PCR results developed recurrent disease, and the pattern of recurrence was similar to that observed in RT-PCR–positive patients. This may arise from the fact that RT-PCR is still suboptimal—some cells may spread via the blood, melanoma cells do not express analyzed molecular markers, and not all melanoma cell transcripts can be detected with our two-marker tests. This last hypothesis may be proven by testing samples obtained from patients with recurring disease by using the two-marker test. From the pathogenic point of view, RT-PCR for tyrosinase/MART-1 mRNA detects melanoma cells regardless of their metastatic potential.4 However, the false-positive rate for prediction of disease relapse is exceptionally low, and even during the relatively short follow-up, we were able to observe disease recurrence in more than 80% of positive patients (only 3 among 18 patients with positive RT-PCR were clinically disease free at the end of follow-up). We are also unable to state the cause for these powerful associations between the RT-PCR assay results and disease outcomes. We may assume that tumor cells in the lymphatics (minimal residual disease) may be the source of subsequent recurrences (both regional and systemic). According to another hypothesis, these cells represent markers of more aggressive/disseminated disease. However, further investigations are necessary to resolve these mechanisms.

In summary, our findings indicate that the use of a two-marker RT-PCR assay for detection of melanoma cells in the lymphatic drainage after radical lymph node dissection in clinically disease-free patients may be a powerful tool in the management of patients with stage III melanoma. We have found a strong relationship between positive two-marker RT-PCR results and early melanoma recurrence, as well as with disease-specific survival time. This test may serve as an early-warning assay for identifying patients who could benefit from more aggressive clinical strategies before the onset of clinically detectable metastatic disease. It may also play an important role in the stratification of stage III melanoma patients for adjuvant treatment protocols, allowing enhanced accuracy in designing study protocols when it is used together with the AJCC staging system of classification.


   ACKNOWLEDGMENTS
 
We thank E. Lorenz for technical assistance and M. Symonides, MD, PhD, for linguistic assistance.


   FOOTNOTES
 
Presented at the Plenary Session of the 57th Annual Cancer Symposium of the Society of Surgical Oncology, March 18–21, 2004.

Preliminary results were presented at the Plenary Session during the 57th Annual Cancer Symposium of the Society of Surgical Oncology, March 18–21, 2004, in New York, NY, USA.

Two-marker reverse transcriptase-polymerase chain reaction assay for tyrosinase and MART1 seems to be useful for the detection of melanoma cells in routinely collected postoperative lymphatic drainage after lymphadenectomy in stage III cutaneous melanoma patients. Positive results with this assay correlate with early recurrence and shorter survival.

Received for publication March 18, 2004. Accepted for publication July 27, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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